Efficient and rapid exact gene replacement without selection

Abstract

We describe a highly efficient method for exact gene replacement in budding yeast. Induction of rapid and efficient recombination in an entire cell population results in at least 50% of the recombinants undergoing a switch of the endogenous copy to a specific mutated allele, with no remaining markers or remnant of foreign DNA, without selection. To accomplish this, a partial copy of the replacement allele, followed by an HO cut site, is installed adjacent to the wild-type locus, in a GAL-HO MATa-inc background. HO induction results in near-quantitative site cleavage and recombination/gene conversion, resulting in either regeneration of wild-type or switch of the endogenous allele to the mutant, with accompanying deletion of intervening marker sequences, yielding an exact replacement. Eliminating the need for selection (over days) of rare recombinants removes concerns about second-site suppressor mutations and also allows direct phenotypic analysis, even of lethal gene replacements, without the need of a method to make the lethality conditional or to employ regulated promoters of unknown strength compared to the endogenous promoter. To test this method, we tried two known lethal gene replacements, substituting the non-essential CDH1 gene with a dominantly lethal version mutated for its Cdk phosphorylation sites and substituting the essential CDC28 gene with two recessively lethal versions, one containing an early stop codon and another inactivating Cdc28 kinase activity. We also tested a gene replacement of unknown phenotypic consequences: replacing the non-essential CLB3 B-type cyclin with a version lacking its destruction box.

Figure 1

Plasmids used in this study. The backbone was RS406 in all cases. A 113-bp HO-site-containing fragment (open box) was inserted at the indicated locations, either downstream of 3′UTR (CDC28 plasmid) or truncating open reading frame to inactivate the (potentially) dominant-lethal coding sequence. The HpaI site and the SmaI site in the 5′ UTR of CDC28 and CLB3 were introduced by mutagenesis, and these sites, and the natural XbaI site in the CDH1 5′UTR, were used for targeting integration.

Figure 2

Synchronous gene conversion of CDC28(mutant)-HO-URA3-CDC28 to CDC28 or CDC28(mutant). MATa-inc GAL-HO strains with the integrated structure shown at bottom were pregrown in synthetic raffinose medium lacking uracil, GAL-HO was induced, and cultures processed as described in Methods. Top left: colonies from single viable cells isolated at the indicated timepoints were grown on YEPD, then assayed for a Ura+ phenotype. Top right: microscopic analysis of microcolonies derived from single cells micromanipulated on YEPD from a CDC28(mutant)-HO-URA3-CDC28 induced for GAL-HO for the indicated time, exhibiting a wild-type morphology, a cdc28-STOP-arrested morphology (few large round cells; see Fig. 3), and a sectored morphology (few large round cells on the edge of a microcolony of wild-type cells). Bottom: PCR analysis of two strains, one with CDC28(STOP) (introduced stop codon marked by a BglII site), and one with CDC28-rsc1 (rsc1 mutation marked with an SfoI site). Middle: PCR with oligos priming in the indicated locations. The right-hand oligonucleotide primes in the HO-cut-site fragment to the left of the HO cut site. Disappearance of product is indicative of processing/loss of DSB-adjacent sequences after HO cleavage. Bottom: the left-hand oligonucleotide primes in two places in the starting construct, in the duplicated CDC28 5′ UTR sequence. The right-hand oligonucleotide primes in sequences in the CDC28 3′ UTR that are not duplicated. Appearance of digestion products (arrows) with BglII or SfoI are indicative of exact gene replacement with the mutant CDC28.

Figure 3

Morphology of CDC28 gene replacements. A: ~18 hrs after galactose induction. cdc28-STOP mutant microcolonies contain ~4–10 large round cells; cdc28-rsc1 cells contain an apparently larger number of extremely hyper-polarized cells. B. 3 days incubation. Both cdc28 mutant replacements are completely inviable: compare microcolony size of a wild-type replacement (top) to the mutants (below). Limited proliferation and/or hyperpolarized bud growth continues for longer in the cdc28-rsc1 microcolonies than the cdc28-STOP microcolonies. C: cdc28-STOP microcolonies, in a wild-type or cdh1 mutant background, were microdissected away from each other and compared to wild-type cells. cdc28-STOP cells are extremely large and round; a minority of cdc28-STOP CDH1 cells are budded, and this budded proportion decreases in a cdh1 background. D: clb2 cdc28-1N synthetic lethality.

Figure 4

Analysis as in Figure 2, with CDH1-m11-delC-HOcs-URA3-CDH1 strains. The extreme hyperpolarized lethal phenotype of CDH1-m11 cells (Robbins and Cross, 2010) is recovered with about 50% efficiency. PCR analysis: top: use of an oligonucleotide priming to the right of the HO cut site shows rapid and efficient cleavage. Bottom: use of an oligonucleotide that is allele-specific to the 9^th phosphorylation site mutation (‘*’) in CDH10-m11 (Zachariae et al., 1998) combined with an oligonucleotide priming outside of the duplicated N-terminal coding sequence of CDH1-m11 demonstrates effective recombination starting at ~4 hrs of galactose induction.

Figure 5

Analysis as in Figure 2, with a CLB3db-delC- HOcs-URA3-CLB3 strain and a CLB3wt-delC- HOcs-URA3-CLB3 control. PCR: top: rapid cleavage of the HO cut site, as in Fig. 5; middle: timecourse of recombination with CLB3db-delC- HOcs-URA3-CLB3 strain, using left-hand allele-specific ‘db’ or ‘wt’ oligonucleotides (priming across the 27-nucleotide db deletion, or within the deleted sequences, respectively), and a right-hand oligonucleotide in C-terminal CLB3 coding sequence not present in the duplicated sequences. Recovery of CLB3-db recombinants started at 2 hrs of galactose incubation; bottom: viable colonies recovered from the CLB3db-delC- HOcs-URA3-CLB3 strain tested for presence of CLB3db or CLB3wt using the same oligonucleotides, demonstrating recovery of pure CLB3-wt or CLB3db colonies respectively. Typically, ~80% of plated single cells were viable, and ~70% of viable colonies contain the CLB3-db mutation. There was no evident growth disadvantage of CLB3-db colonies compared to wild-type. Further characterization of CLB3-db will be reported elsewhere.